Two features make the tooth an excellent model in the study of evolutionary innovations: the relative simplicity of its structure and the fact that the major tooth-forming genes have been identified in eutherian mammals. To understand the nature of the innovation at the molecular level, it is necessary to identify the homologs of tooth-forming genes in other vertebrates. As a first step toward this goal, homologs of the eutherian amelogenin gene have been cloned and characterized in selected species of monotremes (platypus and echidna), reptiles (caiman), and amphibians (African clawed toad). Comparisons of the homologs reveal that the amelogenin gene evolves quickly in the repeat region, in which numerous insertions and deletions have obliterated any similarity among the genes, and slowly in other regions. The gene organization, the distribution of hydrophobic and hydrophilic segments in the encoded protein, and several other features have been conserved throughout the evolution of the tetrapod amelogenin gene. Clones corresponding to one locus only were found in caiman, whereas the clawed toad possesses at least two amelogenin-encoding loci.

A recent landmark paper demonstrates the unique contribution of marsupials and monotremes to comparative genome analysis, filling an evolutionary gap between the eutherian mammals and more distant vertebrate species.

The present review outlines studies of electrophsyiological organization, cortical architecture and thalmocortical and corticocortical connections in monotremes. Results of these studies indicate that the neocortex of monotremes has many features in common with other mammals. In particular, monotremes have at least two, and in some instances three, sensory fields for each modality, as well as regions of bimodal cortex. The internal organization of cortical fields and thalamocortical projection patterns are also similar to those described for other mammals. However, unlike most mammals investigated, the monotreme neocortex has cortical connections between primary sensory fields, such as SI and VI. The results of this analysis lead us to pose the question of what monotremes can tell us about brain evolution. Monotremes alone can tell us very little about the evolutionary process, or the construction of complex neural networks, as an individual species represents only a single example of what the process is capable of generating. Perhaps a better question is: what can comparative studies tell us about brain evolution? Monotreme brains, when compared with the brains of other animals, can provide some answers to questions about the evolution of the neocortex...

This is a summary of the current knowledge of sensory receptors in skin of the bill of the platypus, Ornithorhynchus anatinus, and the snout of the echidna, Tachyglossus aculeatus. Brief mention is also made of the third living member of the monotremes, the long-nosed echidna, Zaglossus bruijnii. The monotremes are the only group of mammals known to have evolved electroreception. The structures in the skin responsible for the electric sense have been identified as sensory mucous glands with an expanded epidermal portion that is innervated by large-diameter nerve fibres. Afferent recordings have shown that in both platypuses and echidnas the receptors excited by cathodal (negative) pulses and inhibited by anodal (positive) pulses. Estimates give a total of 40,000 mucous sensory glands in the upper and lower bill of the platypus, whereas there are only about 100 in the tip of the echidna snout. Recording of electroreceptor-evoked activity from the brain of the platypus have shown that the largest area dedicated to somatosensory input from the bill, S1, shows alternating rows of mechanosensory and bimodal neurons. The bimodal neurons respond to both electrosensory and mechanical inputs. In skin of the platypus bill and echidna snout, apart from the electroreceptors...

Early studies of the echidna led to the conclusion that this monotreme did not have rapid eye movement (REM) sleep. Because the monotremes had diverged from the placental and marsupial lines very early in mammalian evolution, this finding was used to support the hypothesis that REM sleep evolved after the start of the mammalian line. The current paper summarizes our recent work on sleep in the echidna and platypus and leads to a very different interpretation. By using neuronal recording from mesopontine regions in the echidna, we found that despite the presence of a high-voltage cortical electroencephalogram (EEG), brainstem units fire in irregular bursts intermediate in intensity between the regular non-REM sleep pattern and the highly irregular REM sleep pattern seen in placentals. Thus the echidna displays brainstem activation during sleep with high-voltage cortical EEG. This work encouraged us to do the first study of sleep, to our knowledge, in the platypus. In the platypus we saw sleep with vigorous rapid eye, bill and head twitching, identical in behaviour to that which defines REM sleep in placental mammals. Recording of the EEG in the platypus during natural sleep and waking states revealed that it had moderate and high-voltage cortical EEGs during this REM sleep state. The platypus not only has REM sleep...

Phylogenetic reconstructions of the β-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult β-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic β-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the “ε-globin” gene in birds is not orthologous to the ε-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto β-globin gene. Here, we report evidence that the early and late expressed β-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto β-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the ε- and β-globin genes of therian mammals arose via duplication of a proto β-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of β-like globin genes that originated via duplication of a proto β-globin gene in the monotreme lineage. This discovery provides evidence that...

The first specimen of platypus (Ornithorhynchus anatinus) that reached Britain in the late 18th century was regarded a scientific hoax. Over decades the anatomical characteristics of these unique mammals, such as egg laying and the existence of mammary glands, were hotly debated before they were accepted. Within the last 40 years, more and more details of monotreme physiology, histology, reproduction and genetics have been revealed. Some show similarities with birds or reptiles, some with therian mammals, but many are very specific to monotremes. The genome is no exception to monotreme uniqueness. An early opinion was that the karyotype, composed of a few large chromosomes and many small ones, resembled bird and reptile macro- and micro-chromosomes. However, the platypus genome also features characteristics that are not present in other mammals, such as a complex translocation system. The sex chromosome system is still not resolved. Nothing is known about dosage compensation and, unlike in therian mammals, there seems to be no genomic imprinting. In this article we will recount the mysteries of the monotreme genome and describe how we are using recently developed technology to identify chromosomes in mitosis, meiosis and sperm, to map genes to chromosomes...

Background: The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital. Results: We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species. Conclusion: Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition...

We have determined the sequence and genomic organization of the genes encoding the cone visual pigment of the platypus (Ornithorhynchus anatinus) and the echidna (Tachyglossus aculeatus), and inferred their spectral properties and evolutionary pathways. We prepared platypus and echidna retinal RNA and used primers of the middle-wave-sensitive (MWS), long-wave-sensitive (LWS), and short-wave sensitive (SWS1) pigments corresponding to coding sequences that are highly conserved among mammals; to PCR amplify the corresponding pigment sequences. Amplification from the retinal RNA revealed the expression of LWS pigment mRNA that is homologous in sequence and spectral properties to the primate LWS visual pigments. However, we were unable to amplify the mammalian SWS1 pigment from these two species, indicating this gene was lost prior to the echidna-platypus divergence ~21 MYA. Subsequently, when the platypus genome sequence became available, we found an LWS pigment gene in a conserved genomic arrangement that resembles the primate pigment, but, surprisingly we found an adjacent (~20 kb) SWS2 pigment gene within this conserved genomic arrangement. We obtained the same result after sequencing the echidna genes. The encoded SWS2 pigment is predicted to have a wavelength of maximal absorption of about 440 nm...

A dentary of the oldest known monotreme, the Early Cretaceous Teinolophos trusleri, has an internal mandibular trough, which in outgroups to mammals houses accessory jaw bones, and probable contact facets for angular, coronoid, and splenial bones. Certain of these accessory bones were detached from the mandible to become middle ear bones in mammals. Evidence that the angular (homologous with the mammalian ectotympanic) and the articular and prearticular (homologous with the mammalian malleus) bones retained attachment to the lower jaw in a basal monotreme indicates that the definitive mammalian middle ear evolved independently in living monotremes and therians (marsupials and placentals).; Thomas H. Rich, James A. Hopson, Anne M. Musser, Timothy F. Flannery, Patricia Vickers-Rich

The basal lineage of monotremes features an extraordinarily complex sex chromosome system which has provided novel insights into the evolution of mammalian sex chromosomes. Recently, sequence information from autosomes, X chromosomes, and XY-shared pseudoautosomal regions has become available. However, no gene has so far been described on any of the Y chromosome-specific regions. We analyzed sequences derived from Y-specific BAC clones to identify genes with potentially malespecific function. Here, we report the identification and characterization of the mediator complex protein gametologs on platypus Y5 (Crspy). We also identified the X-chromosomal copy which unexpectedly maps to X1 (Crspx). Sequence comparison shows extensive divergence between the X and Y copy, but we found no significant positive selection on either gametolog. Expression analysis shows widespread expression of Crspx. Crspy is expressed exclusively in males with particularly strong expression in testis and kidney. Reporter gene assays to investigate whether Crspx/y can act on the recently discovered mouse Sox9 testis-specific enhancer element did reveal a modest effect together with mouse Sox9+Sf1, but showed overall no significant upregulation of the reporter gene. This is the first report of a differentiated functional male-specific gene on platypus Y chromosomes...

Ghrelin is a growth hormone (GH)-releasing and appetite-regulating peptide predominately released from the stomach. Ghrelin is evolutionarily highly conserved and known to have a wide range of functions including the regulation of metabolism by maintaining an insulin-glucose balance. The peptide is produced as a single proprotein, which is later proteolytically cleaved. Ghrelin exerts its biological function after O-n-octanoylation at residue serine 3, which is catalyzed by ghrelin O-acyl transferase (GOAT) and allows binding to the growth hormone secretagogue receptor (GHS-R 1a). Genes involved in the ghrelin pathway have been identified in a broad range of vertebrate species, however, little is known about this pathway in the basal mammalian lineage of monotremes (platypus and echidna). Monotremes are particularly interesting in this context, as they have undergone massive changes in stomach anatomy and physiology, accompanied by a striking loss of genes involved in gastric function. In this study, we investigated genes in the ghrelin pathway in monotremes. Using degenerate PCR, database searches and synteny analysis we found that genes encoding ghrelin and GOAT are missing in the platypus genome, whilst, as has been reported in other species...

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall...

Olfactory cues are thought to play a crucial role in the detection of the milk source at birth in mammals. It has been shown that a marsupial, the tammar wallaby, can detect olfactory cues from its mother's pouch at birth. This study investigates whether the main olfactory and accessory olfactory system are similarly well developed in other marsupials and monotremes at birth/hatching as in the tammar. Sections of the head of various marsupial and two monotreme species were investigated by light microscopy. Both olfactory systems were less well developed in the kowari and Eastern quoll. No olfactory or vomeronasal or terminal nerves could be observed; the main olfactory bulb (MOB) had only two layers while no accessory olfactory bulb or ganglion terminale were visible. All other investigated marsupials and monotremes showed further developed olfactory systems with olfactory, vomeronasal and terminal nerves, a three-layered MOB, and in the marsupials a prominent ganglion terminale. The main olfactory system was further developed than the accessory olfactory system in all species investigated. The olfactory systems were the least developed in species in which the mother's birth position removed most of the difficulty in reaching the teat...

Genetic sex determination systems are generally based on the presence of differentiated sex chromosomes. Birds have a ZZ/ZW sex chromosome system in which males are ZZ and females ZW, whereas mammals have an XX/XY system with males being XY and females XX. Monotremes have an extraordinary sex chromosome system that consists of multiple sex chromosomes: 5X5Y in platypus and 5X4Y in echidna. Intriguingly, the monotreme sex chromosomes show extensive homology to the bird ZW and not to the therian XY. However, sex determination in monotremes is still a mystery; the Y-specific Sry gene that triggers male sex determination in therian mammals is absent and so far very few genes have been identified on Y chromosomes in monotremes. To gain more insights into the gene content of Y-chromosomes and to identify potential sex determination genes in the platypus a collaborative large scale transcriptomic approach led to the identification of new male specific genes including the anti-Muellerian hormone AMH that I mapped to Y₅, this makes Amhy an exciting new candidate for sex determination in monotremes. Platypus chromosome 6 is largely homologous to the therian X and therefore it represents the therian proto sex chromosome. In addition, this autosome features a large heteromorphic nucleolus organizer region (NOR) and associates with the sex chromosomes during male meiosis (Casey and Daish personal communication). I investigated chromosome 6 heteromorphism in both sexes and found a number of sex-specific characteristics related to the extent of the NOR heteromorphism...

Background: Major histocompatibility complex (MHC) class I genes are found in the genomes of all jawed vertebrates. The evolution of this gene family is closely tied to the evolution of the vertebrate genome. Family members are frequently found in four paralogous regions, which were formed in two rounds of genome duplication in the early vertebrates, but in some species class Is have been subject to additional duplication or translocation, creating additional clusters. The gene family is traditionally grouped into two subtypes: classical MHC class I genes that are usually MHC-linked, highly polymorphic, expressed in a broad range of tissues and present endogenously-derived peptides to cytotoxic T-cells; and non-classical MHC class I genes generally have lower polymorphism, may have tissue-specific expression and have evolved to perform immune-related or non-immune functions. As immune genes can evolve rapidly and are subject to different selection pressure, we hypothesised that there may be divergent, as yet unannotated or uncharacterised class I genes. Results: Application of a novel method of sensitive genome searching of available vertebrate genome sequences revealed a new, extensive sub-family of divergent MHC class I genes, denoted as UT...

The first specimen of platypus (Ornithorhynchus anatinus) that reached Britain in the late 18th century was regarded a scientific hoax. Over decades the anatomical characteristics of these unique mammals, such as egg laying and the existence of mammary glands, were hotly debated before they were accepted. Within the last 40 years, more and more details of monotreme physiology, histology, reproduction and genetics have been revealed. Some show similarities with birds or reptiles, some with therian mammals, but many are very specific to monotremes. The genome is no exception to monotreme uniqueness. An early opinion was that the karyotype, composed of a few large chromosomes and many small ones, resembled bird and reptile macro- and micro-chromosomes. However, the platypus genome also features characteristics that are not present in other mammals, such as a complex translocation system. The sex chromosome system is still not resolved. Nothing is known about dosage compensation and, unlike in therian mammals, there seems to be no genomic imprinting. In this article we will recount the mysteries of the monotreme genome and describe how we are using recently developed technology to identify chromosomes in mitosis, meiosis and sperm, to map genes to chromosomes...